5-Fluorouracil (5-FU) was first introduced in 1957 and remains a mainstay in treatment of solid tumors including colorectal cancer (CRC). 5-FU exerts cytotoxic activity primarily through inhibition of thymidylate synthase (TS) and to some extent also through incorporation of metabolites into RNA. (Ford et al. (2002) Clinical Cancer Research 2002; 8(1): 103-109). The overall response rate of 5-FU alone is quite limited (10-15%) and modulation strategies have been developed to increase the anticancer activity of 5-FU. (Johnston et al. Anticancer Drugs 2001, 12: 639-646). One of the most widely used strategies involves the co-administration of the folate 5-formyl tetrahydrofolate (folinic acid or leukovorin or LV) with 5-FU. (Romanini et al. (1991) Cancer Res., 51: 789-793; Keyomarsi et al. (1988) J. Biol. Chem., 263: 14402-14409). LV stabilizes the ternary complex that inhibits thymidylate synthase (TS), an enzyme necessary for DNA synthesis. (Longley et al. (2003) Nat. Rev. Cancer, 3(5):330-8). By adding LV to 5-FU the overall response rates increased to over 20%. (Id.).
A reduced folate, fotrexorin calcium (CoFactor®) ((dl)-5,10,-methylenepteroyl-monoglutamate calcium salt, or [6R,S]-5,10-methylene-THF Ca salt), also known as racemic methyleneTHF, has been suggested as an alternative to LV based on the assumption that direct administration of the reduced folate methyleneTHF in place of LV might offer significant advantages with respect to clinical activity. CoFactor® is a 1:1 mixture of the two diastereoisomers (Odin, E., Carlsson, G., Frösing, R., Gustaysson, B., Spears, C. P., Larsson, P. A., 1998. Chemical stability and human plasma pharmacokinetics of reduced folates. Cancer Invest. 16, 447-455). As the [6R]-isomer is the directly active co-substrate of TS, it was anticipated that the administration of CoFactor®, instead of leucovorin, would be advantageous due to lower inter- and intrapatient variability regarding both clinical safety and efficacy.
Indeed, in a Phase II Trial in previously untreated metastatic colorectal cancer, the response rate for CoFactor® was found to be 35% (Saif, M. W, Merritt, J, Robbins J, Stewart J., Schupp, J, 2006. Phase III Multicenter Randomized Clinical Trial to Evaluate the Safety and Efficacy of CoFactor®/5-Fluorouracil/Bevacizumab Versus Leucovorin/5-Fluorouracil/Bevacizumab as Initial Treatment for Metastatic Colorectal Carcinoma Clinical Colorectal Cancer, Vol. 6, No. 3, 229-234, 2006), and in another phase I/II clinical trial it was demonstrated that CoFactor® combined with 5-FU showed clinical benefit in pancreas cancer, defined as stable disease or tumor response, in 40% of patients (Saif, M. W., Makrilia N., Syrigos K., 2010. CoFactor: Folate Requirement for Optimization of 5-Fluouracil Activity in Anticancer Chemotherapy. Journal of Oncology Vol. 1-5). However, apart from presenting an unnecessary hepatic detoxification burden, the unnatural (6S)-isomer is a partial competitive inhibitor of the natural [6R]-isomer regarding its effect as co-substrate for TS (Leary, R. P., Gaumont, Y., Kisliuk, R. L., 1974. Effects of the diastereoisomers of methylenetetrahydrofolate on the reaction catalyzed by thymidylate synthetase. Biochem. Biophys. Res. Commun. 56, 484-488). Furthermore, in a Phase IIb study CoFactor® in colorectal cancer was not demonstrated to be more efficacious than leucovorin as no significant differences between the study arms with regard to either efficacy or safety could be found, and a planned Phase III study colorectal cancer was discontinued before completion (Press release: ADVENTRX Provides Update on Cofactor Program. Nov. 2, 2007).
There remains a great need for compositions and methods for stabilizing the ternary complex and enhancing the inhibition of TS. The inventors have surprisingly discovered that administration of 6R-MTHF increases plasma levels of 2′-deoxyuridine (dUrd) compared to the administration of equimolar concentrations of LV. Surprisingly, the inventors have discovered that administration of 6R-MTHF increases the inhibition of TS compared to the administration of equimolar concentrations of LV.